Application of REP-PCR in genotyping of Candida glabrata

Abstract

Objective To investigate the role of repetitive extragenic palindromic-PCR(REP-PCR) in the genotyping of Candida glabrata.Methods Thirty-four Candida glabrata isolates from patients with deep fungal infection were collected,and the biochemical phenotypes of isolates were identified by API 20C AUX yeast identification strip.Genotyping of 34 Candida glabrata isolates was performed with REP-PCR.Fungal genome DNA was extracted,primers for PCR was designed from Care-2 repetitive elements,the products of PCR were analysed by electrophoresis,and cluster analysis was conducted with NTSYS software.Isolates with the same genotype had a similarity index(SI) ≥97% and no significant band difference,and those with SI ≥97% and one band difference were characterized as subtypes.Indexes of discriminatory power(DP) were compared between REP-PCR genotyping and multilocus sequence typing(MLST),and the REP-PCR genotyping of Candida glabrata with different biochemical phenotypes was analysed.Results REP-PCR genotyping revealed there were 17 genotypes for these 34 Candida glabrata isolates,including type A(8 isolates),type B(6 isolates),type C and D(3 isolates for each type),type E(2 isolates) and type F to Q(1 isolate for each type),and there was no subtype.DP(95% CI) of genotyping by REP-PCR and MLST were 0.911(0.770-0.980) and 0.369(0.220-0.560) respectively,and there were significant differences between them(χ2=21.68,P0.01).There were two biochemical phenotype codes for these 34 Candida glabrata isolates,which were 2000040(32 isolates) and 6000040(2 isolates),and the latter two isolates were classified into type D and type K in REP-PCR genotyping(SI=0.43).Conclusion REP-PCR is simple to operate and is powerful in genotyping of Candida glabrata,which is suitable for the epidemiological study of Candida glabrata in clinical laboratories.